The first aim is to investigate the role of cysteine residues in CFTR Cl channel gating. This work will build on preliminary data which shows that reducing or oxidizing agents and cvsteine modifying reagents change the activity of and gating characteristics of the CFTR channel. Experiments outlined in this proposal will focus on mutagenesis of the cysteine residues in various combinations up to all of them. Replacing cysteines with non-reactive residues can highlight those that are involved in channel gating and those which mediate the effects of cysteine- modifying reagents. The second aim of this proposal is to exploit the reactivity of cysteine residues to investigate structure- function relationships of CFTR using a cysteine-less mutant. Cysteine residues will be reintroduced iii to the sequence at key points and the mutants will be tested for sensitivity to cysteine modification. By investigating changes in CFTR gating which occur when cysteines are located at different sites, it should be possible to learn more about the role of the nucleotide binding domains, the R domain and to find regions which are important for forming the pore or the vestibule of the channel. Understanding how redox-sensitive residues modulate channel gating may be important clinically as anti-oxidant drugs are currently prescribed to CF patients as mucolytic agents. Redox state may be an important regulator of CFTR channel activity in cells, while learning more about the structure and function of CFTR could provide for new and better treatment options.